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Multi-Parametric Imaging of Hypoxia and Cell Cycle in Intestinal Organoid Culture

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Multi-Parametric Live Cell Microscopy of 3D Tissue Models

Part of the book series: Advances in Experimental Medicine and Biology ((AEMB,volume 1035))

Abstract

Dynamics of oxygenation of tissue and stem cell niches are important for understanding physiological function of the intestine in normal and diseased states. Only a few techniques allow live visualization of tissue hypoxia at cellular level and in three dimensions. We describe an optimized protocol, which uses cell-penetrating O2-sensitive probe, Pt-Glc and phosphorescence lifetime imaging microscopy (PLIM), to analyze O2 distribution in mouse intestinal organoids. Unlike the other indirect and end-point hypoxia stains, or point measurements with microelectrodes, this method provides high-resolution real-time visualization of O2 in organoids. Multiplexing with conventional fluorescent live cell imaging probes such as the Hoechst 33342-based FLIM assay of cell proliferation, and immunofluorescence staining of endogenous proteins, allows analysis of key physiologic parameters under O2 control in organoids. The protocol is useful for gastroenterology and physiology of intestinal tissue, hypoxia research, regenerative medicine, studying host-microbiota interactions and bioenergetics.

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Acknowledgments

This work was supported by Science Foundation Ireland (SFI) grants 13/SIRG/2144 (RID) and 12/RC/2276 (IAO and DBP).

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Authors and Affiliations

  1. Laboratory of Biophysics and Bioanalysis, School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland

    Irina A. Okkelman & Dmitri B. Papkovsky

  2. Department of Anatomy and Neuroscience, University College Cork, Cork, Ireland

    Tara Foley

  3. Metabolic Imaging Group, School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland

    Ruslan I. Dmitriev

Authors
  1. Irina A. Okkelman
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  2. Tara Foley
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  3. Dmitri B. Papkovsky
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  4. Ruslan I. Dmitriev
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Correspondence to Ruslan I. Dmitriev .

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Editors and Affiliations

  1. Metabolic Imaging Group, School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland

    Ruslan I. Dmitriev

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Okkelman, I.A., Foley, T., Papkovsky, D.B., Dmitriev, R.I. (2017). Multi-Parametric Imaging of Hypoxia and Cell Cycle in Intestinal Organoid Culture. In: Dmitriev, R. (eds) Multi-Parametric Live Cell Microscopy of 3D Tissue Models. Advances in Experimental Medicine and Biology, vol 1035. Springer, Cham. https://doi.org/10.1007/978-3-319-67358-5_6

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